Methods for amplifying a target nucleic acid sequence that may be present in a test sample are, by now, well known in the art. Such methods include the polymerase chain reaction (PCR) which has been described in U.S. Pat. Nos. 4,683,195 and 4,683,202, the ligase chain reaction (LCR) described in EP-A-320 308, gap LCR (GLCR) described in European Patent Application EP-A-439 182, multiplex LCR described in International Patent Application No. WO 93/20227 and the like. These methods have found widespread application in the medical diagnostic field as well as the fields of genetics, molecular biology and biochemistry. Unfortunately, one drawback of nucleic acid amplification reactions is that they are mostly qualitative.
The nature of amplification reactions makes it difficult for them to be used to quantitatively detect the presence of a target sequence which may be present in a test sample. Accordingly, while traditional amplification reactions are useful for detecting the presence of a minute quantity of a target sequence in a test sample, traditional amplification reactions generally cannot be employed to determine the quantity of a target sequence in a test sample. However, variations of traditional amplification reactions have been developed which enable quantitative amplification reaction analysis.
One quantitative amplification reaction is called "competitive amplification." This method is commonly applied to PCR. According to competitive amplification reactions, a standard nucleic acid sequence competes with a target sequence during the amplification reaction. Generally, the standard sequence and a sample suspected of containing a target sequence are combined in a dilution series in which the amount of the standard sequence is constant in all members of the series. Alternatively, the standard sequence and sample sequence are combined in a dilution series in which the amount of standard sequence is varied among the members of the dilution series. In any case, the concentration of the standard sequence in the members of the dilution series, is known. PCR is then performed on all members of the dilution series and results in the production of a mixture of two nucleic acid sequence species. One species derived from the standard sequence and one species derived from the sample sequence. The concentration of each species in a particular dilution depends on the number of copies of the standard and sample sequences in the dilution prior to amplification. During the amplification reaction, detectable groups are typically introduced into both types of sequences. After amplification, the two species are separated and the amount of detectable group incorporated into each species is determined. This detection procedure is performed for each member of the dilution series. A competition curve can then be generated and the amount of sample sequence can be extrapolated based on the known amounts of standard sequence.
Methods of competitive amplification have been described in U.S. Pat. No. 5,219,727; Kinoshita T., et. al., Analytical Biochemistry 206: 231-235 (1992); and Jalava T., et. al., BioTechniques15:(1), 134-205 (1993). While these competitive amplification techniques have shown utility, they require substantial amounts of sample preparation as well as technician interaction and a concomitant risk of sample contamination. In addition, the use of a standard sequence adds an additional reagent not generally a requirement of traditional amplification reactions. Moreover, performing amplification on members of a dilution series requires more reagents than performing amplification on a single sample. All of these factors add to the costs of performing competitive amplification reactions.
Another quantitative amplification reaction is "kinetic amplification analysis". This method takes advantage of a dye's ability to bind double stranded nucleic acid sequences. For example, PCR generally produces double stranded nucleic acid sequences. In the presence of a dye, such as ethidium bromide, which binds double stranded nucleic acid sequences, an increase in fluorescence is observed with successive rounds of PCR amplification. The greater the amount of target nucleic acid sequence in a test sample, the earlier a rise in fluorescence will be observed.
Kinetic amplification analysis has been described in Higuchi R., et. al., Bio/Technology 11: 1026-1030 (1993). Unfortunately, the efficiency of an amplification reaction can vary from sample to sample. Hence, while two samples may contain equivalent target sequence concentrations, different fluorescent rates for the two samples may be obtained in a kinetic amplification analysis. Accordingly, this method is not always useful in a clinical setting because of the wide variety of samples which are assayed.
Thus there is a need for a method of quantitatively performing an amplification reaction which does not require excess technician manipulation or reagents, and can be employed in a clinical laboratory setting.